A genetic epilepsy syndrome called GEFS+ (Generalized Epilepsy with Febrile Seizures plus) is now known to be a common form of genetic epilepsy. At least three of the identified GEFS+ mutations occur in the gene encoding the gamma2 subunit of the GABA A receptor, responsible for most synaptic inhibition in the brain. One such mutation, gamma2(R43Q), replaces a highly conserved arginine residue with a glutamine. Molecular modeling predicts that this substitution may disrupt salt bridge interactions between the gamma2 and beta2 subunits, which may be critical for normal receptor function and pharmacology. Our initial studies of human gamma2(R43Q)-containing GABA A receptors reveal dramatic kinetic changes, including enhanced receptor desensitization, that could contribute to GEFS+ by causing a "rundown" in synaptic inhibition during high intensity neural activity. The overall Objective of the proposed research is to understand how alterations in the region of the GABAA receptor near gamma2(R43Q) change receptor function and contribute to epilepsy. The Specific Aims are to 1) determine the biophysical mechanism of the changes in receptor function caused by the mutation, by studying channel kinetics in excised membrane patches, 2) determine the structural basis of the changes by examining point mutations designed to a) rescue the predicted salt bridge formation and b) to further purturb it in controlled manner, 3) determine the impact of the gamma2(R43Q) mutation on synaptic transmission in transgenic mice that express the mutant subunit in place of the wild type allele, and 4) determine whether synapses undergo enhanced desensitization as a result of the mutation, and whether this causes increased network hyperexcitability in cortical, thalamic and hippocampal brain slices. The knowledge gained from these studies will 1) provide valuable information about the structural/functional role of the receptor region affected by the epilepsy mutation gamma2(R43Q), 2) allow us to better understand critical aspects of inhibition that are causally related to epilepsy, 3) provide an unprecedented opportunity to study the physiological role of GABA A receptor desensitization at functional synapses and in network excitability; and 4) further develop a novel animal model for future studies of GEFS+ epilepsy.